| Salmonella enterica serovar Enteritidis(SE)is the most common etiological agent of food-borne salmonellosis,leading to intestinal or systemic diseases in humans and poultry.The AI-2/LuxS system is widely distributed in gram-negative and gram-positive bacteria which regulates a range of behaviors in diverse bacteria,including biofilm formation,virulence-associated traits,and antibiotic susceptibility.Although LuxS system has been identified in S.enteritis,its effects on biological characteristics of SE are not clear.Therefore,this paper investigates the effect of LuxS QS system on growth features,biofilm formation as well as antimicrobial susceptibility of SE through construction of deletion of luxS gene required for AI-2 synthesis in LuxS QS system.This study preliminarily explores the effects of AI-2/LuxS system on biological characteristics of SE.To investigate the effect of AI-2/LuxS system on biological characteristics of SE,the ?luxS mutants were constructed by using the lambda Red homologous recombination method.The luxS deletion mutants were complemented by cloning parent strains luxS gene along with the native promoter,into the low copy number vector pWKS30.The recombinant plasmid(pWKS30-luxS)was sequenced to confirm the correct insert,and was transformed by electroporation into their isogenic luxS mutant to obtain the complementary strain C-SM32?luxS.To study the effect of LuxS system on biological characteristics of SE,?luxS gene stability in ?luxS mutant was analyzed,and its growth features,adherence and invasion,as well as biofilm formation were determined and compared with the wild type.The ?luxS mutant was serially passaged 25 times on antibiotic-free agar medium;the ?luxS gene was not restored to wild-type luxS gene.Growth curve measurements were determined at different times,showing that the ?luxS mutant grew slightly faster than the parent strains in vitro.To investigate the difference in biofilm formation between the parental and the mutant strains under the same culture conditions,biofilm formation was quantitatively analyzed using microtiter plate assay for different time periods(24,48,and72h).Biofilm structures were observed using confocal laser scanning microscopy(CLSM),and expression of biofilm-related genes was investigated using RT-PCR.The results showed that both the parental and the mutant strains formed more biofilms with increasing time.The biofilms formed by the luxS mutant strain were higher than that by the wild-type strain at 24 h,48h,and 72 h.The CLSM images reveal that the luxS mutant strain tends to aggregate into distinct clusters and relatively dense structures,whereas the parental strain appears to be confluent and more evenly distributes.The expression levels of biofilm-related genes csgA?ompR?csgB and csgD in the parental and mutant strains were determined by qRT-PCR.Compared with the parental strain,the csgA and omp R expression was significantly up-regulated in the mutant strain,whereas no obvious difference was detected in the csgB and csgD expression.Adhesion and invasion of the parental and the mutant strains to Caco2 cells and DF-1 cell in vitro showed that the ?luxS mutant had significantly increased adhesion and invasive ability to Caco-2 and DF-1 cell lines compared with the parental strain.The expression levels of virulence-related genes sopB,sseL,mgtC,and fimA in the parental and mutant strains displayed that compared with the parental strain,the sopB,sseL,mgtC,and fimA expression was significantly up-regulated in the mutant strain.This result suggests that LuxS system affects adhesion and invasion of SE.Furthermore,antimicrobial susceptibility test showed that deletion of luxS resulted in increasing susceptibility(128-to 256-fold)to quinolones and 2-to 8-fold to ampicillin,tetracycline,doxycycline,chloramphenicol,and florfenicol,but no changes in the susceptibility was found to amikacin and gentamicin.This finding suggests that LuxS system affects antimicrobial susceptibility of SE to certain antimicrobials.In summary,the above results showed that deletion of lux S does not affect SE growth rate,but increases antimicrobial susceptibility,biofilm formation and adhesion and invasion to Caco-2 cells and DF-1 cells.The findings provides a basis for the further study on AI-2/LuxS system in the regulation of behaviors in SE. |
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